supplementary information hdac mediated suppression of ... · shy1 spbc1215.02c ucp8 spbc83.02c...

Supplementary Information HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin Ozan Aygün, Sameet Mehta and Shiv I. S. Grewal Supplementary Figures 1-8

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2565

FLAG-IP

Input

WB:H3K4me2

H3-FLAG:

H3-FLAG:

WB:H3K4me2

17kDa

17kDa

emosoel cu

N ycnapucco

) 2gol (

2

-22

-2

0

0

1 kb

pht1

shy1

SPBC1215.02c

ucp8

SPBC83.02c tas3

apc15 SPBC83.05

SPBC83.06c

jmj3 rvb2

Supplementary Figure 1 Replication-independent H3 replacement occurs at nucleosome-depleted regions associated with gene promoters across euchromatin. (a) H3 turnover measured by MNase-ChIP (ChIP/Input) is plotted in alignment with a 20kb region of chromosome 2. Names of the open reading frames given above or below correspond to genes transcribed from forward or reverse strands, respectively. Nucleosome occupancy (as described in Fig.1) across the same euchromatic domain is plotted in the bottom panel for comparison. The sites of high H3 replacement and nucleosome depletion associated with gene promoters are highlighted with pink shading. (b) Functional incorporation of FLAG tagged histone H3 into chromatin. Immunoprecipitation (IP) and western blotting (WB) experiment to detect the incorporation of H3-FLAG into chromatin. Extracts from the indicated strains were used to perform immunoprecipitation with anti-FLAG antibody prior to Western blot analyses with an antibody that recognizes H3 di-methylated at lysine 4 (H3K4me2).

a

b

H3 re

plac

emen

t: M

Nase

–ChI

P en

richm

ent (

log2

)


imr2R dh dg

clr4∆ / WT

swi6∆chp2∆ / WT

nmt-epe1 / WT

dcr1∆ / WT

ago1∆ / WT

ago1∆epe1∆ / WT

epe1∆ / WT

chp2∆ / WT

swi6∆ / WT

clr3∆ / WT

1234

1234

1234

1234

1234

1234

1234

1234

1234

1234

2.70

2.76

2.71

2.50

1.90

1.47

1.49

1.77

1.38

2.10

Supplementary Figure 2 Quantitation of the increase in H3 replacement across the pericen-tric heterochromatin in mutants as compared to wild-type cells, normalized to euchromatin. Average increase in histone H3 turnover observed in each mutant across the heterochromatin was calculated as described in the top panel. The fold change in H3 turnover values normalized to euchromatin is presented at the right describing the fold changes for each mutant.

gua1klp5

Foldhistone H3 turnover

H3 turnover (Mutant)

H3 turnover (WT)

H3 turnover (Mutant)

H3 turnover (WT)

heterochromatin euchromatin


b Heterochromatin1kbimr2R dh dg tRNA

-3

0

3

-3

0

3

a

clr4∆

WT

H3-FLAG

Tubulin

clr4∆WT

0 15 45 120 0 15 45 120Time after H3-FLAG

induction (min):

c

clr3∆

WTclr4∆

ycnapucco 3H enot si H

P en

richm

ent

I hC- 3H-i t na

3

2

1

0imr2R dh dg tRNA

Supplementary Figure 3 Changes in H3 replacement are not due to ectopic H3-FLAG levels or gross differences in endogenous H3 occupancy. (a) Expression of H3-FLAG was monitored by anti-FLAG western blotting 15, 45, and 120 minutes after shifting cells into growth medium containing sucrose. Detection with anti-Tubulin is used as loading control. (b) H3 exchange was measured in wild-type and clr4∆ strains as described in Fig. 1a, except cells were crosslinked 15 minutes after H3-FLAG induction. (c) Effect of loss of Clr4 or Clr3 on histone H3 occupancy after synchronization by hydroxyurea (HU). Total (endogenous) histone H3 occupancy was measured in the same parental WT, clr3∆ and clr4∆ strains used in H3-replacement experiments, except the cells do not express any H3-FLAG. Cells were grown into mid-exponential phase and synchronized by hydroxyurea as described in Fig.1. ChIP analysis was performed by using anti-histone H3 antibody and analyzed by tiling microarray. The ChIP-on-chip data (ChIP/Input) is plotted across the right pericentromeric repeats of chromosome 2.

H3

repl

acem

ent:

MN

ase–

ChI

P en

richm

ent (

log2

)

1kb


clr4∆

dcr1∆

ago1∆

WT1234

1234

1234

1234

clr4∆

Relative fold expression:

WT WTdcr1∆

swi6∆

chp2∆

chp2∆swi6∆

clr3∆

H3-FLAG

Tubulin

epe1

∆

ago1

∆epe

1∆

ago1

∆

nmt-e

pe1

0.04 1.0 0.02 1.01.6 2.2 1.5 0.6 0.99 0.82 1.2 4.0 1.2 0.3

Sucrose Sucrose

esoculG

esoculG

a

b

imr2R dh dg

Histone H3 ChIP under H3-FLAG inducing conditions

imr2R dh dg

Normalized H3 turnover H3 turnover / Total H3

Supplementary Figure 4 The increased H3 turnover in heterochromatin mutants is not due to increased H3 occupancy. (a) Comparison of induction levels of H3-FLAG in the strains used in H3 replacement experiments. Lysates prepared from the indicated strains were analyzed by SDS-PAGE and western blotting with anti-FLAG and anti-Tubulin antibodies. Relative fold change in expression of H3-FLAG as compared to WT are shown. (b) Cells expressing H3-FLAG, which are grown exactly as described in Fig. 1a, were used to measure H3 occupancy by ChIP-chip using anti-H3 antibody. Total histone H3 levels (left panel) and H3 turnover values normalized to the H3 occupancy (right panel) are plotted across the pericentromeric heterochromatin domain.


3

-3

0

3

-3

0

3

-3

0

clr4∆

WT

dcr1∆

scr1

SPBC1D7.01 end3

mrt4 trs20 rpa34

sme1

mtr10 lcf1 tif35fis11 kb

Supplementary Figure 5 Loss of Clr4 or Dcr1 does not significantly alter H3 replacement patterns across euchromatic loci. H3 replacement measured across the 33kb long euchromatic region in chromosome 2 using MNase-ChIP-on-Chip analysis as described in Fig. 1a. Open reading frames are shown in alignment with H3 replacement as described in Supplementary Fig. 1a.

H3

repl

acem

ent:

MN

ase–

ChI

P en

richm

ent (

log2

)


b

cdc25–22 clr4∆

cdc25–22

Heterochromatin1kbimr2R dh dg tRNA

-5

0

5

-5

0

5

cIR-Rmat3MIR-L mat2P 1kb

Heterochromatin

-5

0

5

-5

0

5 cdc25–22

Supplementary Figure 6 Analysis of replication-independent histone H3 exchange across the heterochromatin domains in G2 arrested cells. (a) Schematic description of the experiment. H3 exchange (ChIP/Input) is measured essentially as described in Fig.1, except instead of using HU and G1–S arrest, here cells are arrested at the G2 phase of the cell cycle by using the temperature-sensitive mutation cdc25–22. (b) H3 exchange across the pericentromeric repeats in G2 arrested cells. H3 exchange was measured in cdc25–22 and clr4∆cdc25–22 cells as described in a. The data is plotted across the right pericentromeric repeats of chromosome 2. (c) H3 replacement at the silent mating–type locus. H3 turnover is analyzed as described in b , except the data is plotted across the silent–mating type region.

a

cells transformed with pINV1–h3.2 –FLAG

grown in glucose at 26 0C

G2 arrest by shifting temperature to 37 0C

4hCells switched to

sucrose medium to induce H3–FLAG

Replication–independent H3–FLAG incorporation

MNase–ChIP

Analysis by tiling microarray

cdc25–22

at 37 0C

30min&

cdc25–22 clr4∆

H3

repl

acem

ent:

MN

ase–

ChI

P en

richm

ent (

log2

)H

3 re

plac

emen

t: M

Nas

e–C

hIP

enric

hmen

t (lo

g2)


cen-dh

act1

-RT

WT clr3∆ clr4∆

a b

WT

clr3∆

clr4∆

-FOA + FOA

Supplementary Figure 7 Heterochromatic silencing defects of the strains used in histone replacement experiments. (a) Reverse transcriptase (RT)-PCR analysis performed with total RNA isolated from the indicated strains using primers that amplify centromeric dh repeats (cen-dh), or act1 RNA as a control. -RT denotes the negative control reaction without reverse transcriptase. (b) Silencing assay performed by spotting 10-fold serial dilutions of the same strains containing the ura4+ reporter gene integrated into outer centromeric repeats. Cells were grown on medium with or without 5-FOA. Cells that can suppress the ura4+ reporter gene can grow in 5-FOA, however the drug is toxic for the cells which can not repress ura4+ expression. (c) RT-PCR analysis performed as described in a, except primers that amplify the indicated regions of the silent mating-type region were used for amplification.

cIR-Rmat3MIR-L mat2P cenH

WT

clr3∆

clr4∆

(mat53) (mat55)

otr1Rotr1L

ura4+

cnt1

imr1Ldgdh imr1R dhdg


imr2R dh dg tRNA cA9K3H

cA41K3H

6

45

3210

6

45

321

0

clr3∆

WTclr4∆

clr3∆

WTclr4∆

IR-Rmat3MIR-L mat2P

0

0.5

1.5

2.5

2

1

cA41K3H

0

0.5

1.5

2.5

2

1

cA9K3H

Supplementary Figure 8 Loss of Clr3 or Clr4 increases histone acetylation across heterochromatin domains. (a) The indicated strains were grown into mid-exponential phase and synchronized by hydroxyurea before crosslinking for ChIP experiments. Histone H3 Lysine 14 (top) or Lysine 9 (bottom) acetylation was measured by ChIP-chip analysis (ChIP/Input) and plotted across the pericentromere. (b) H3K9 and H3K14 acetylation were measured exactly as described in a. The data is plotted across the silent mating type locus.

a

b

clr3∆

WTclr4∆

clr3∆

WTclr4∆

ChIP

enr

ichm

ent

ChIP

enr

ichm

ent

ChIP

enr

ichm

ent

ChIP

enr

ichm

ent


supplementary information hdac mediated suppression of ... · shy1 spbc1215.02c ucp8 spbc83.02c...

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